Abstract It has been long known that protein kinase A (PKA) activated by cAMP is essential for sperm motility and fertility. Targeted disruption of the sperm specific catalytic subunit of PKA (C?2) or the soluble adenylyl cyclase (sAC) results in male infertility. Sperm from both these knock out mice have impaired motility and lack the ability to undergo capacitation and hyperactivation required for fertilization. While the role of PKA in sperm function is well established little is known about the protein substrates of PKA. Determination of the identities of these proteins will enable us to understand mechanisms underlying sperm motility and fertility. We will use a novel chemical-genetic approach to identify sperm PKA substrates. This approach uses a genetically engineered modification of the ATP binding domain of the catalytic subunit of PKA such that the modified enzyme recognizes specific ATP analogues that are not accepted by other kinases. Phosphorylation of substrates in the presence of the ATP analog enables their identification with relative ease. We have, in hand, the knock-in mice harboring the modified gene for the catalytic subunit of PKA and mice with a targeted disruption of the sperm sAC. We are generating double mutant mice expressing the analog sensitive PKA on a sAC null background. We propose two specific aims. In the first aim sperm proteins from the analog sensitive knock-in mice and the double-mutant mice will be phosphorylated with an ATP analog. Phosphorylated proteins will be detected by analog-specific antibodies followed by their identification using tandem MS. In the second aim we will confirm that the proteins are valid PKA substrates and determine how changes in their phosphorylation occur during sperm maturation in the epididymis and during sperm activation events required for fertilization. Successful execution of these aims should lead to a mechanistic understanding of how cAMP and PKA regulate sperm function.